Advances in tumor immunology have led to a better understanding of the immunoinhibitory mechanisms (e.g. inhibitory ligands/receptors and regulatory T cells) that create a major barrier to effective anti-tumor immunity. One of the most critical pathways responsible for mediating tumor-induced immune suppression is the programmed death-1 (PD-1) pathway. While the early results seen with PD-1 pathway blocking antibodies (Abs) in cancer have been encouraging, further study is required to optimize their use in RCC patients. Preliminary correlative studies suggest that while tumor PD-L1 expression may increase the likelihood of benefit with anti-PD-1, it fails to identify all responders. Predictive biomarker development and validation will help to guide this approach to the proper patients (Aims 1 and 2). The application of PD-1 blockade in the treatment nave setting may yield even better results and obviate the need for subsequent lines of therapy (Aim 2). Using a multi-faceted approach, this project will provide a better understanding of the mechanisms of action and resistance to PD-1 pathway blockade, thereby facilitating the development of effective combination regimens (Aims 2 and 3). We will study the impact of intra-tumoral heterogeneity on biomarker development through our unique collection of matched primary tumor and metastasis specimens in our SPORE Tissue Bank and samples obtained from two prospective clinical trials of patients receiving anti-PD-1 antibodies. Laboratory experiments will integrate novel Abs, engineered proteins and genetic tools including: 1) anti-PD-1, PD-L1, and PD-L2 monoclonal Abs for IHC of human tissues and long term treatment of mice; 2) whole-exome and transcriptome sequencing of both tumor and infiltrating T cells (TILs), which can be used to explore mechanisms of innate resistance, and 3) innovative murine models such as mice that conditionally lack PD-1 on specific cell types and reporter mice that enable FoxP3 visualization. This highly collaborative team of basic and clinical scientists brings together an exceptional collective expertise in RCC immunotherapy trials, the PD-1 pathway and tumor immunoregulation, which should facilitate the generation of clinically meaningful results.